CN101381409A - N-terminally chemically modified protein compositions and methods - Google Patents

Abstract

Provided herein are methods and compositions relating to the attachment of water soluble polymers to proteins. Provided are novel methods for N-terminally modifying proteins or analogs thereof, and resultant compositions, including novel N-terminally chemically modified G-CSF compositions and related methods of preparation. Also provided is chemically modified consensus interferon.

Description

Protein composition that N-is terminally chemically modified and method

The application is to be February 8 nineteen ninety-five the applying date, and application number is 95191454.5, and denomination of invention is divided an application for the application for a patent for invention of " protein composition that N-is terminally chemically modified and method ".

Invention field

The present invention generally speaking relates to the protein modification field, and, more particularly relate to protein and analogue thereof are adhered to neck city (term used herein " protein " and " polypeptide " or " peptide " these terms of water soluble polymerizer, as do not do other explanation and all refer to the same meaning). the invention still further relates to the novel method that is used for the terminal modified protein of N or its analogue and the composition of gained. on the other hand, the present invention relates to terminally chemically modified G-CSF composition of new N and relevant preparation method. the invention still further relates to the Interferon, rabbit that has of chemically modified.

Background of invention

At present; why treatment can obtain with suitable form and liberal quantity with protein. and the operability of recombinant protein causes the progress of protein formulations and chemically modified aspect. and one of purpose of this chemically modified is exactly a protein protection. and chemical attachment can stop contacting of proteolytic ferment and protein skeleton itself effectively, thereby has stoped proteinic degraded.Other advantage is included under the certain environmental conditions, increasing proteinic stability of treatment and cycling time and reduce its immunogenicity. the review article of describing protein modification and fused protein is: Francis Focus on Growth Factors, 3:4-10 (in May, 1992) is (by Mediscript, Morntview Court, Friern Barnet Land, London N20, OLD, UK publishes).

Polyoxyethylene glycol (" PEG ") is exactly a kind of this class chemically modified part, it is used for preparation treatment with proteinaceous product (verb " PEGization " (pegylate) mean adhere at least one PEG molecule). and for example Adagan is the PEG modified forms of adenosine deaminase, has been got permission to be used for the treatment of serious composite immune deficiency disorders; The sudismase of PEGization has entered the clinic trial stage of treatment head injury; The alpha-interferon of PEGization is tested in the I clinical trial phase of treatment hepatitis; Also entered the preclinical test stage according to another the report glucocerebrosidase of PEGization and the methemoglobinemia of PEGization. adhere to polyoxyethylene glycol to the proteoclastic provide protection (Sada that appears in the newspapers; Deng J.Fermentation Bioengineering 71:137-130; 1991); the method of adhering to specific polyalkylene glycol moiety is referring to U.S. Patent No. 4; 179; 337; Davis et al.; " non-immunogenicity polypeptide "; announce on December 18th, 1979. and U.S. Patent No. 4; 002,531, Royer; " polyoxyethylene glycol is to the product of the modification and the production thereof of enzyme "; if announced on January 11st, 1977. wanting has more fully understanding to this field, can be referring to " Enzymes as Drugs " such as Abuchowski (J.S.HolcerbergandJ.Roberts, eds.pp.367-383 page or leaf (1981)).

Other water soluble polymerizer that has used such as ethylene glycol, propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone gathers-1,3-dioxolane (poly-1,3-dioxolane), gather-1,3, the 6-trioxane, the EMA anhydride copolymer, polyamino acid (or single aggressiveness or random copolymer).

For peg molecule, can it be added on the protein by several different methods.Generally speaking, peg molecule is to be linked on the protein molecule at the reactive group of finding on the protein molecule by one. amino, as on the lysine residue or the N-end on amino be generally used for this class and adhere to.For example, Royer (U.S. Patent number 4,002,531, see above-mentioned) think and can utilize standard reductive alkylation reaction that peg molecule is attached on the enzyme .Wright in disclosed EP 0 539 167 on April 28th, 1993, think in " Peg Imidates and ProteinDerivates Thereof " that the peptide and the organic double compound that have free amino group can be modified by the direct derivative of PEG or relevant water-soluble organic polymer.Shaw relates to the number that is used for adhering to by the reactive amino group lysine residue of peg molecule in the modifying protein in February 27 nineteen ninety in the laid-open U.S. Patents numbers 4,904,584.

" G-CSF ", granulocyte colony-stimulating factor are a kind of concrete treatment through chemically modified and induce the fast breeding of neutrophilic granulocyte with protein .G-CSF and be discharged in the blood, thereby the treatment effect is provided in anti-infective.

The open EP 0 401 384 of European patent has described material and the method that is used to prepare the G-CSF that is attached with peg molecule on December 12 nineteen ninety disclosed being entitled as " granulocyte colony-stimulating factor of chemically modified ".

Report G-CSF and the analogue thereof modified among the EP 0 473 268 of on March 4th, 1992 disclosed being entitled as " continuity discharges and comprises a water-soluble polymeric medicinal compositions on the peptide molecule covalent attachment ", described the use of the derivative that water-soluble granular a polymer such as a polyoxyethylene glycol produced on various G-CSF and the covalent attachment thereof.

Has an active polypeptide of human myelomonocyte colony-stimulating factor in what on October 4th, 1989, disclosed EP 0 335 423 reported a kind of modification.

Another example is the IL-6 of PGEization, is being entitled as " hIL-6 of modification " (referring to common unsettled U.S.S.N.07/632,070). EP 0 442 724 in disclose peg molecule be attached on the IL-6 molecule.

Disclosed EP 0 154 316 reported that the aldehyde of a lymphokine (Lymphokine) and a polyoxyethylene glycol reacts on September 11st, 1985.

Yet relating to, many methods of adhering to polymer to protein molecule use certain part as spacer group. these parts often have antigenicity. can carry out a kind of tresylchloride method that does not relate to spacer group, but because the use of tresyl chloride can produce malicious byproduct, so be difficult to this method production for treating product.Referring to Francis etc.: the stability of pharmaceutical grade protein: the strategy of intravital degradation pathway and protein stabilization (Eds.Ahern., T.and Manning, M.C.) (plenum, New York, 1991).Simultaneously also can be referring to Delgado etc., the eds. such as .Fisher that " activate coupling PEG to the application of protein in the immune affinity cell preparation " by Tresyl chloride, the water system is separated in cytobiology and Application in Biotechnology, Plenum Press, N.Y.N.Y., 1989 pp.211-213.

Chamow etc. (Bioconjugat ē Chem.5:133-140 (1994)) have reported that CD4 immunosorption element (immanoadhesin) is by modifying with mono methoxy poly glycol aldehyde generation reductibility alkanisation. the author reports that 50% CD4-Ig carries out the MePEG modification under the controlled condition of PEGization level of response.Ibid, sees 137 pages.The author has also reported external, the CD4-Ig that modifies is negative correlation in conjunction with the ability of (to albumen gp120) and the degree of its MePEGization. and ibid, equally also can be referring to Rose et al., Bioconjugate Chemistry 2:154-159 (1991) has reported in the literary composition that linking group carbohydrazide (carbohydrazide) optionally is attached on the C-terminal carboxyl(group) of protein substrate (Regular Insulin).

Yet, in whole field or relate in the field of specified protein and also do not have a kind of method can allow water soluble polymerizer optionally be attached on the N-end of protein such as G-CSF. What is more, the method that exists provides non-selective attachment reaction on any reactive group at present, no matter these groups be in protein (for example, the lysine side-chain group), still on the N-end. this will cause the heterogeneity of product. for example, the G-CSF molecule of PEGization, contain the polyalkylene glycol moiety that number is different from other molecule in some molecule. in above-mentioned reaction, may form a kind of heterogeneity mixture such as a molecule that contains 5 lysine residues, some molecules contain 6 peg molecules, have 5 in some molecules, have 4 in some molecules, have 3 in some molecules, have 2 in some molecules, and have 1 even a peg molecule and also do not have. and in having the molecule of some polyoxyethylene glycol, peg molecule may not can in different molecules be attached to identical position.

More than these all be exploitation treatment with the PEGization proteinaceous product not in sharp factor. when developing this class medicine, product is bioactive predictablely to seem exceptionally important. for example, superoxide-dismutase non-selectively with peg molecule bonded situation under, the enzyme that a part is modified completely lose activity (P.McGoff et al.Chem.Pharm.Bull.36:3079-3091 (1988)) if. these therapeutic proteins each between-lot in composition is had any different, people can't predict its biological activity. different loci bonded peg molecule stability is different, and this situation may cause these molecules to dissociate out from protein.Certainly, if these molecules be adhere at random and therefore RANDOM SOLUTION from, treatment just can not accurately be predicted with proteinic pharmacokinetics so. consider that from human consumer's angle be not quite similar different cycling times in batches, so also may be inaccurate on the dosage.Consider that from the producer's angle treatment of getting permission to come into the market will increase trouble with proteinic preservation. in addition, the aforesaid method none provides the selective N-terminal chemically modified of no connection portion (between protein and the polymer).If share the connection portion, because possible antigenicity may have a negative impact.

Therefore, still needing can the terminally chemically modified protein of selective N and analogue, comprises that method the present invention of G-CSF and total Interferon, rabbit (following is that example is illustrated with the protein of these two kinds of chemically modifieds just) has set forth this needs from all many-sides.

The present invention's general introduction

The present invention relates to N-terminally chemically modified proteinic basically all-goods and used method. unexpectedly the G-CSF of the terminal chemically modified of N-demonstrates the undiscovered advantage aspect stable of all kinds of institutes of other G-CSF that contains a chemically modified on another site of this molecule. more beat all is present method at the G-CSF that is used for preparing the chemically modified of N-end, find to use the standard reductive alkylation reaction that the condition of optionally modifying the N-end can be provided, and this method can be widely used in other protein (or its analogue) and G-CSF. surprisingly, utilize the standard reductive alkylation reaction, its end product-with amine key and water-soluble polymer bonded protein, much more stable than identical polymer/protein connector with amido linkage (amide link age). the protein that another one is modified like this (following just described as embodiment) is to have Interferon, rabbit (consensus interferon).Therefore, will describe in detail as top, the present invention has the content of all many-sides of the special modification that relates to chemically modified protein matter (or its analogue) and specified protein.

On the one hand, the present invention relates to the goods and the method for homogeneous basically of the G-CSF (or its analogue) of the terminal chemically modified of N-. the G-CSF of mono-PEG that the terminal single PEGization of N-has been described among the embodiment below is more stable than the G-CSF of the single PEGization of other type. in addition, since the N-end of G-CSF molecule in reaction process easily and peg molecule approaching, the terminal PEGization ratio of N-is higher, so this method operates and relatively has advantage.

The invention still further relates to a kind of standard reductive alkylation reaction type. this reacts the amino group of activator matter optionally or its analogue N-terminal residue, thereby make water-soluble polymer part selectivity be attached to the N end. provide goods and (if using polyoxyethylene glycol) of the homogeneous basically of polymer/protein bound molecule to have the goods that polyalkylene glycol moiety directly is coupled to the PEGization protein molecule on the protein portion like this. this method that is used for G-CSF and total Interferon, rabbit is described below, and these contents provide others of the present invention.

The accompanying drawing summary

Figure 1A is-the color atlas replica at the G-CSF ion-exchange chromatography peak of PEGization.

The SDS-PAGE figure of the single PEGization G-CSF of Figure 1B all kinds.

Fig. 2: line A is the SEC-HPLC figure of recombinant human methylenedisulfonyl G-CSF standard; Line B is the SCM-PEG-GCSF reaction mixture; Line C is the G-CSF of the terminal PEGization of N-; Line D is the G-CSF of the single PEGization of 35 Methionins; Line E is the G-CSF. of Methionin 41 single PEGization

Fig. 3 A, 3B and 3C are the G-CSF of the terminal PEGization of N-for the figure of the HPLC after endoproteinase (endoproteinase) SV8 peptase is cut, 3A; 3B is the G-CSF of the single PEGization of 35 Methionins; 3C is the G-CSF. of Methionin 41 single PEGization

Fig. 4 is a bar graph, be shown in external, the G-CSF kind of single PEGization and the not bioactive comparison of standard of PEGization.

Fig. 5 A and 5B have shown the bioactive analytical results of G-CSF derivative of in vivo single PEGization, Fig. 5 A shows the G-CSF of hamster through the terminal PEGization of subcutaneous injection N-, the mean value of white count behind the G-CSF of the G-CSF of the single PEGization of 35 Methionins or Methionin 41 single PEGization, Fig. 5 B show the final average white blood cell count area under curve behind the G-CSF of the above-mentioned various single PEGization of the single subcutaneous injection derivative.

Fig. 6 A, 6B and 6C scheme for the SEC-HPLC of the stable gained of the G-CSF of the G-CSF of the terminal PEGization of research N-or the single PEGization of 35 Methionins. and Fig. 6 A is at pH6.0, the SEC-HPLC figure of the G-CSF stability of the terminal single PEGization of research N-under 4 ℃ of conditions, Fig. 6 B are the G-CSF figure of the single PEGization of 35 Methionins under the same terms. and Fig. 6 C is presented under pH6.0 and 4 ℃ further the G-CSF results of stability to the single PEGization of 35 Methionins. and the time (" T ") is represented the sky.

Fig. 7 has shown that screen analysis (size exclusion) the HPLC analytical results of reaction mixture in the process of standard reductive alkylation reaction takes place for rh-G-CSF and methoxy polyethylene glycol aldehyde (MW6kDa).

Fig. 8 has shown reaction mixture screen analysis (size exclusion) the HPLC analysis chart with MPEG (also at MW=6kDa) N-hydroxyl succinyl-ester.

Fig. 9 is single N-terminal M PEG-GCSF joiner (the MPEG aldehyde by the rh-G-CSF different molecular weight makes through the reductibility alkylation reaction, and wherein the molecular weight of MPEG is 6kDa, 12kDa and 20kDa) total white cell response situation in back of single subcutaneous injection dosage.

Describe in detail

The present invention relates to the goods of basically homogeneous of the terminal chemical modifying protein of N-and used Method.

On the one hand, the present invention relates to the composition of G-CSF of N-end chemical modification and used method thereof.

This method (be used for the end modified G-CSF of N-and standard reductive alkylation reaction method) provides the mixture of the basically homogeneous of single aggressiveness/protein attachment. and " basically homogeneous " used herein means each polymer of observing/protein attachment molecule and all only contains a polymer moieties. perhaps can comprise the unreacted protein of (namely lacking polymer moieties) in the goods. among the embodiment below, confirm by peptide mapping (peptide mapping) and N-end sequencing, at least contain single aggressiveness of 90%/protein attachment in the goods of preparing, the unreacted protein content is no more than at most 10%. preferably, the terminal single PEG compound matter content of N-should reach 95% (as below described in the embodiment) of goods at least, most preferably, the terminal single PEG compound matter of N-account for goods 99% or higher. this single aggressiveness/protein attachment has biologically active. the homogeneity that the G-CSF goods of the terminal PEGization of the N-of " basically homogeneous " provided herein mean these goods is enough to demonstrate the superiority that the homogeneous goods have, for example, be easy to the different pharmacokinetics in batches of prediction in clinical practice.

People can select to prepare polymkeric substance/protein connector molecule mixture, advantage provided herein is, people can select the single aggressiveness/ratio of protein connector in mixture. like this, if necessary, people just can prepare the range protein mixture of the polymer molecule that has different numbers and adhere to (such as 2,3,4 or the like), and combine with the single aggressiveness/protein connector that utilizes the inventive method to prepare, just can obtain the mixture of booking list aggressiveness/protein connector ratio.

The embodiment that uses G-CSF is provided below; As mentioned above; G-CSF is the treatment protein that is used for the treatment of hematopoietic disorders. in general; G-CSF useful in the invention process can separate from the mammal organism; The product of chemical synthesis process as selecting perhaps; Or the product that obtains by prokaryotes or the exogenous dna sequence dna of eucaryote host expresses; Wherein these DNA can obtain from genome or cDNA clone or by DNA is synthetic. suitable prokaryotes host comprise various bacteriums (asE. Coli); Suitable eukaryote host comprise yeast (as S. Cerevisiae) and mammalian cell (as: Chinese hamster ovary cell, MC). according to used host's difference, the G-CSF expression product may be by the carbohydrate institute glycosylation in Mammals or other eukaryotic cell, or also can comprise an initial methionine residues (at the-1) in the non-glycosylated .G-CSF expression product. though the G-CSF that preferably recombinates (particularly from E.coli), the present invention have predicted and have used arbitrarily or whole maximum commercial practicality of this class form of G-CSF in other biology.

Existing some G-CSF analogues of report have biological function, and these molecules also can be chemically modifieds, for example, and one or more peg molecules in the interpolation.The G-CSF analogue is in U.S. Patent No. 4,810, existing report in 643.The tool report has the example of bioactive other G-CSF analogue that AU-A-76380/91 is arranged, EP 0 459 630, EP 0 272703, described in EP 0 473 268 and the EP 0 335 423 those, though the activity of the disclosed every kind of analogue of report is not provided in the literary composition. also can be referring to AU-A-10948/92, PCTUS 94/00913 and EP 0 243 153.

In general, G-CSF and analogue thereof useful among the present invention can be by carrying out chemically modified program provided herein optionally to modify the terminal alpha-amino group of N-, the required biological property of test products obtained therefrom, biological activity test for example provided herein is determined. certainly, if desired non-human mammal is carried out this class treatment, can use the inhuman G-CSF of reorganization, as the G-CSF product of the mouse of recombinating, ox and dog etc. for example, referring to PCT WO 9105798 and PCT WO 8910932.

Therefore, another aspect of the present invention comprises the composition of the G-CSF analogue of the terminal chemically modified of N-. as mentioned above, the G-CSF analogue can comprise have aminoacid addition, disappearance and/or substitute (with the G-CSF aminoacid sequence described in the following embodiment 1 comparatively speaking) product.Time expection performance function is for being attached on the G-CSF acceptor and the analogue of non-essential N-end with the G-CSF analogue of the generation of selective stimulating neutrophil leucocyte by PEGization when the N-end for those, see Hill et al., PNAS-USA 90:5167-5171 (1993); Also can be referring to PCT US 94/00913.

The polymer molecule that uses can be selected from water-soluble polymer.(for reductive alkylation process on glycopeptides described herein, polymer should have single reaction aldehyde radical). selective polymerisation thing molecule should be water miscible, make it want the bonded protein molecule in water surrounding, deposited phenomenon can not take place, for example in the physiological environment. consider from standard reductive alkylation reaction angle, the polymer molecule of selecting should have a reaction aldehyde radical, so just can control extent of polymerization according to the inventive method. and polymer can be a branch or not branched.Therepic use for the end product goods, preferred polymerizable molecular should be medicinal acceptable. whether those skilled in the art should be able to make in treatment according to this polymkeric substance/protein connector is used for selecting required polymkeric substance, if in treatment, use, then should consider its dosage, cycling time is to proteoclastic resistibility and some other factor.For G-CSF, the available means of testing that provides is herein found out, those skilled in the art also should select suitable means of testing at other therapeutic protein. and water-soluble polymer is freely optional: top listed (background parts) various molecules and dextran or poly-(n-V-Pyrol RC), polyoxyethylene glycol, polypropylene glycol, polyoxytrimethylene/ethylene oxide (prolypropylene oxide/ethylene oxide) multipolymer, polyoxyethylene polyvalent alcohol and polyvinyl alcohol.

Consider that for optimization described below the molecular weight of polymkeric substance can be any size, and can be to have branch or not branched.For polyoxyethylene glycol, most preferred molecular weight is to be about 2kDa to (term " approximately " means in the polyoxyethylene glycol goods, and some molecules are greater than described molecular weight, and other molecules are less than described molecular weight) between about 100kDa.The following examples 1 and embodiment 2 relate to PEG6000, its selection is convenient to purifying and a suitable modular system is provided. and required treatment plan (the slowly-releasing time length that situation is for example required is depended in the use of other size, effectively talk about aspect biological activity if render a service, the accessibility of handling, antigenicity size or shortage and some other PEG are to the known effect of therapeutic protein and analogue thereof.

A concrete aspect of the present invention is exactly the G-CSF molecule of the terminal single PEGization of N-, it partly is made up of a polyalkylene glycol moiety and a G-CSF. for said composition, people can be from multiple peg molecule (with molecular weight and have or not branch etc.), peg molecule is to the ratio of G-CSF protein molecule in the reaction mixture, the type of the PEGization reaction of being carried out, the aspects such as type of the method for the G-CSF of acquisition selective N-terminal PEGization and the G-CSF that will use are selected.In addition, this composition and method also comprise the prescription of medicinal compositions, the treatment of medicament and production method.

Peg molecule is the same with their concentration in reaction mixture to the ratio of protein molecule, can change. in general, optimum proportion (is meant reaction efficiency, be not have unnecessary unreacted protein and polymer molecule in the reaction mixture) will measure according to selected molecular weight polyethylene glycol. in addition, the purifying of the terminal single PEGization product of the non-specific PEGization reaction that relates among the embodiment as present method and N-subsequently, ratio depend on the available reactive group number that can participate in reaction (be typically ∝ or

Amino). the protein that relates among the embodiment of this paper: the reaction ratio of PEG molecule is quite low to obtain to be essentially the product (each protein molecule is to 1.5 PEG molecules) of single PEGization.

In order to obtain the G-CSF of the terminal PEGization of N-, also there is multiple PEGization the reaction method aforesaid or standard reductive alkylation described in the embodiment 2 reaction below available. about the polymerization process of no linking group between polyalkylene glycol moiety and the protein portion exists at Franciset al: the stability of protein medicine: vivo degradation approach and make the strategy (Eds.Ahern. of protein stabilization, T.and Manning, M.C.Plenum, New York, 1991) had description in. in addition, Delgado et al., " by TresylChloride Activiation method coupling PEG to the application of protein in the immune affinity cell product " and Fisher et al., eds., the water system is separated in cytobiology and Application in Biotechnology, Plenum Press, N.Y.N.Y., relate among 1989 pp.211-213 and utilize tresyl chloride, there is not linking group to exist between polyalkylene glycol moiety and protein portion thereby make. this method is owing to producing malicious byproduct, so be difficult to the production for treating product.Relating to the N-hydroxyl succinyl-fat that uses carboxymethyl methoxy polyoxyethylene glycol in an example of the present invention. another embodiment as following lecture in more detail relates to use standard reductive alkylation reaction method.

Obtain the method (that is, if necessary, this part being separated from the part of other single PEGization) of the G-CSF goods of the terminal PEGization of N-can be in the G-CSF of the PEGization of associating molecule colony the product of purifying N-end PEGization.For example, in the following embodiments, the G-CSF of PEGization at first separates to obtain to have the single PEGization material (material that may have other a plurality of PEGization that contain identical apparent charge) of charge characteristic by ion-exchange chromatography, use sieve chromatography (size exclusion chromatography) to separate the product of single PEGization then. by this method, the G-CSF of the terminal single PEGization of N-to be separated from the material of the material of the single PEGization of other type and other a plurality of PEGization. other method is appeared in the newspapers. for example, PCT WO 90/04606, reported a kind of method of the PEG-of separation protein adduct mixture in (May 3 nineteen ninety is open), comprising separating (partitioning) protein adduct with the aqueous two phase system PEG/ that contains PEG.

On the other hand, the present invention also provides selectivity to obtain the method for the terminal chemically modified protein matter of N-(or its analogue). and following for a kind of standard reductive alkylation that utilizes reacts the method for carrying out protein modification, it utilizes obtainable monovalence amino (primary amino group) the dissimilar specific reaction of (Methionin is to N-terminal) to carry out the modification of specified protein.Under appropriate reaction conditions, successfully proteinic N-end has been carried out optionally modifying with the carbonyl that has a polymkeric substance. under the pH that can utilize epsilon-amino and protein N-terminal alpha-amino pKa difference on the lysine residue, carry out this reaction. by this selective modification, the control water-soluble polymers is attached on the protein: the connection of polymkeric substance mainly occurs in proteinic N-end, and other reactive group (as the side chain amino on the lysine residue) is not then significantly modified and taken place.

Even more important and surprisingly, the invention provides a kind of method of goods of the homogeneous basically for preparing single aggressiveness/protein connector molecule, need not use required being further purified of other chemically modified chemical process. in addition, product with amine key is more stable than the product that forms with amido linkage unexpectedly. during will studying in great detail below, this point obtains proof. more particularly, if use polyoxyethylene glycol, the present invention also provides does not have the antigenicity of possibility linking group, and has the polyalkylene glycol moiety that directly is coupled to protein portion and the protein of the terminal PEGization of N-of nontoxic byproduct.

But the reaction See Figure is described (sodium cyanoborohydride is the illustrative reductive agent among the figure):

Therefore, an aspect of of the present present invention content is the method for preparing polymkeric substance/protein connector, comprise: (a) under the standard reductive alkylation reaction conditions, to contain protein molecule and water-soluble polymers molecular reaction more than an amino, the pH of reaction solution should be suitable for selecting to activate the aminoterminal alpha-amino group of said protein molecule, so described water-soluble polymers is attached to described alpha-amino group group, and; (b) obtain reaction product. can from optional from unreacted molecule reaction product isolated, but for treatment with product, this is preferred.

Another aspect of the present invention content is this standard reductive alkylation reaction, it can optionally be attached to polymer molecule arbitrarily has on the alpha-amino protein at the N-end, and providing the goods of the homogeneous basically of a kind of single aggressiveness/protein connector. terminology used here " single aggressiveness/protein connector " means by a polymer moieties and is attached to the composition of forming on the protein portion (those connectors that also comprise use protein analogue as herein described). and single aggressiveness/protein connector has a polymer moieties that is positioned at (as the amino of lysine side-chain) on N-end rather than the side chain amino. and goods preferably contain the single aggressiveness/protein connector greater than 80%, the single aggressiveness/protein connector more preferably greater than 95%.

For the single aggressiveness/protein connector molecule colony of basic homogeneous, its reaction conditions should be to be attached on the N-end of desired protein with allowing the water-soluble polymers molecular selectivity.This reaction conditions generally causes the amino and terminal alpha-amino pKa difference of N-(pK is meant amino protonated and non-protonization respectively accounts for 50% o'clock pH) of Methionin. in general, for different protein, can use different pH so that the terminal alpha-amino group of N-obtains best the modification.

PH often also can influence the ratio of used polymer molecule and protein molecule. in general, if reaction solution pH is lower than pK, the polymer molecule ratio should substantially exceed protein molecule and be advisable that (that is: the alpha-amino group reactivity of N-end is few more, it is many more to reach the required polymkeric substance of top condition) if. pH is higher than pK, polymkeric substance then: the ratio of protein molecule unnecessary too big (reactive group that promptly obtains is many more, and required polymer molecule is few more).

Another essential condition that need consider is the molecular weight of polymkeric substance. and in general, the molecular weight of polymkeric substance is big more, and the polymer molecule number that is attached on the protein is few more. and same, when optimizing these parameters, whether polymer molecule has branch also should take in.In a word, polymericular weight high more (or branch is many more), polymkeric substance: proteinic ratio also should be high more.

Standard reductive alkylation reaction hereto, its reductive agent should be more stable at the aqueous solution, preferably only can reduce at formed western Buddhist alkali of reductibility alkylation reaction starting stage (Schiffbase). preferred reductive agent can be selected from sodium borohydride, sodium cyanoborohydride, boric acid dimethylamine, boric acid Trimethylamine 99 and boric acid pyridine. what use among the embodiment below is sodium cyanoborohydride.

The water-soluble polymers molecule can be above-described type, should possess single reaction aldehyde radical in its molecule is coupled on the protein molecule it. for polyoxyethylene glycol, use is used for being used for G-CSF coupling and PEG12000 and has Interferon, rabbit being coupled at and describing below. what deserves to be mentioned is for G-CSF, in present method, PEG 12000,20000 and 25000 were all successfully used. and the polyoxyethylene glycol propionic aldehyde (is seen U.S. Patent No. 5,252,714) stability has certain advantage owing to it has preferably in water.

As mentioned above, present method is widely used in having terminal alpha-amino any protein of N-and analogue thereof. for example, the proteinaceous product that obtains by the exogenous DNA of bacterial expression, because the result of bacterial expression, have an alpha-amino group on the methionine(Met) of its N-end (methionyl) residue. as mentioned above, comprise peptide, all be some peptide analogs and other modifying protein. protein analogue, the total Interferon, rabbit that aforesaid G-CSF analogue and non-natural exist all is applicable to this method.

Therefore, for the G-CSF of the terminal chemically modified of this N-, any G-CSF as herein described or its analogue all share (article that for example, ibid described those).Used among the following embodiment is the reorganization G-CSF that produces in the bacterium, have 174 amino-acid residues and an extra dl-methionine residue. as described herein, chemically modified can use any water-soluble polymers molecule described herein to carry out, and what use in the following examples is polyoxyethylene glycol.

Total Interferon, rabbit is an another kind of protein used in the present embodiment. it is following that what will describe is the single PEGization modification of carrying out protein N-end with the reaction of the reductibility alkanisation among the present invention, thereby prepare the total Interferon, rabbit of chemically modified. therefore, another aspect of the present invention relates to these goods. as used herein, joint owner's lymphocyte interferon is called " total Interferon, rabbit " at this, or " IFN-con ", meaning is a non-natural polypeptide, it mainly comprises those amino-acid residues that all natural human LeIF hypotype sequences are total, and it also is included in all hypotypes and does not have on common amino acid whose one or more positions, mainly betide the amino acid in this position, and under any circumstance, be not included in the arbitrary amino acid residue .IFN-con that does not come across this position at least one naturally occurring hypotype and comprise three aminoacid sequences, difference called after IFN-con1, IFN-con2 and IFN-con3, the United States Patent (USP) 4 that it is being owned together, 695,623 and 4,897, open in 471, this paper quotes it in full for your guidance. (United States Patent(USP) Nos. 4,897,471 and 4,695, use " α " name in 623, and this paper does not use). the dna sequence dna of coding IFN-con can synthesize the .IFN-con polypeptide by above-mentioned patent or other standard method and preferably pass through conversion or transfection synthetic dna sequence dna in host bacterium, especially E.coliExpress and produce. be that IFNcon is reorganization IFN-con.With method known to those skilled in the art with at Klein et al., J.Chromatog. 454: 205-215 (1988) but in the method purifying that is used for IFN-con1 described preferably exist E.coliIn the IFN-con of the IFN-con. purifying produced may comprise its mixture of isomers, as including first sulfonyl IFN-con1 among the IFN-con1 of purifying, that removes methionyl IFN-con1 and N-end closure removes methionyl IFN-con1 (Klein et al., Arc.Biochem.Biophys. 276: 531-537 (1990)).As selection, IFN-con can comprise the isomer of specific isolation, available isomeric form of coming IFN-con separated from one another such as the technology of isoelectric focusing well known by persons skilled in the art.

Therefore, another aspect of the present invention content is the total Interferon, rabbit of chemically modified, wherein said total Interferon, rabbit is to be selected from IFN-con1, IFN-con2, and during the IFN-con3. chemically modified with water-soluble polymers as herein described PEG for example, and the reductive alkylation process on glycopeptides that is used for the terminal selective chemical modification of N-. the embodiment 3 of this paper has described the chemically modified of IFN-con1, comprises the IFN-con1 molecule is combined with peg molecule (PEG12000) at the N-end.

On the other hand, the PEGization protein that the inventive method is produced is that polyalkylene glycol moiety directly is connected with protein portion, there is not spacer group to exist, do not produce any malicious byproduct yet. comprise G-CSF and total Interferon, rabbit as herein described among the embodiment. for the colony of PEGization G-CSF protein molecule, wherein polyalkylene glycol moiety is directly to be coupled to (but might not be the colony of the G-CSF molecule of the terminal PEGization of N-) on the G-CSF protein portion, and the standard reductive alkylation reaction can be carried out in acid or nonacid environment.

A further aspect of the present invention is about above-mentioned relevant medicinal compositions. this medicinal compositions can be suitable for injection, or it is oral, act on using of lung, nasal cavity and other form. in general, be contained in medicinal compositions among the present invention and comprise the of the present invention single aggressiveness/protein connector product and the medicinal acceptable diluent that goes up of significant quantity, preservatives, solubilizing agent, emulsifying agent, adjuvant and/or carrier.Comprise various damping fluid components (as: Tris-HCl, acetate, phosphoric acid salt) in this based composition, the thinner of pH and ionic strength; Additive such as stain remover and solubilizing agent (as: Tween 80, poly sorbitol ester 80), antioxidant (as xitix, sodium metabisulfite), preservatives (as: merthiolate, phenylcarbinol) and filler (as: lactose, N.F,USP MANNITOL); These materials are mixed polymer molecule such as poly(lactic acid) (polylacticacid), in the particulate state goods that polyoxyethylene glycol acid etc. is made or mix in the liposome. these compositions may influence the proteinic physical condition stability of the terminal chemically modified of N-of the present invention, removing speed in release rate in vivo and the body. for example referring to Remington ' s " pharmacology " the 18th edition.(1990, Mack publishing company., Easton, PA18042) pp.1435-1712. this paper quotes for your guidance.

The present invention has an aspect to relate to the treatment and the production method of medicine again. use that polymkeric substance/G-CSF connector that the present invention produces (or have natural G-CSF the active analogue of short hemopoietic) relaxes or the characteristic feature of the symptom nursed one's health is, going down of hematopoiesis or immunologic function, and the minimizing of the neutrophil leucocyte number of more specifically saying so. these symptoms are perhaps because (for example chemotherapy or radiotherapy) causes in other lysis of treatment. this also may be owing to suffer from infectious diseases, such as bacterium, virus, fungi or some other catch. for example, because septicemia due to the infectation of bacteria.Perhaps, this symptom is caused by heredity or environmental factors, reduces disease or leukemia as the severe chronic neutrophilic granulocyte. and age factor is also very important, and in presbyatrics, patient's neutrophil number can reduce or the neutrophil reactivity reduces.Some these class symptoms are seen Filgrastim (r-met Hu G-CSF) in clinical Practice, Morstyn, G. reaching T.M.Dexter compiles., Marcel Dedder Inc., N.Y. (1993), pp.351. some other research is few through using that polymer of the present invention/G-CSF connector relaxes and the symptom of regulating also has the minimizing of lipid in the blood (or cholesterol), and some cardiovascular symptoms are because G-CSF can induce the generation plasminogen activator.Up to now, the enable mode of G-CSF (or analogue) in these situations is also not fully aware of.The interpolation of water-soluble polymers molecule (as polyoxyethylene glycol) bring benefit can for the patient use, because the G-CSF frequency injection that bioactive postpone retention can allow each course of treatment is still less.

In general, be the symptom of those application of interference elements but use the symptom that polymkeric substance/total Interferon, rabbit can be alleviated and nurse one's health that the present invention produces, comprise cell generation disorders, virus infection and autoimmunity disorder are as comprehensive angiosclerosis.With reference to McManus Balmer, DICP, " pharmacological agent yearbook " 24:761-767 (1990) (application of the modifying factor of biologically active in cancer therapy: look back. first part, Interferon, rabbit). method and composition thereof with total interferon therapy cell generation disorders can be referring to disclosed PCT WO92/06707 on April 30th, 1992, this paper quotes for your guidance. and for example, the total interferon molecule of the PEGization that the available the present invention of hepatitis (A, B, C, D, E type) produces is treated.Prove in the following embodiments that external the total interferon activity of chemically modified is 20%. of the total Interferon, rabbit non-chemically modified

Above-mentioned various molecules, owing to carried out further research, thereby the data of used suitable dose level when having obtained the treatment of symptoms different among the relevant different patient. those of ordinary skill is being considered treatment plan, after patient age and the healthy state, can determine proper dosage. in general, should between every kg body weight 0.01 μ g to 100 μ g, (only calculate the weight of protein portion, not have calculational chemistry to modify the weight of part) for injection or perfusion dosage.

The following examples will be mentioned each side more than will illustrating. in embodiment 1, the G-CSF by the terminal PEGization of N-compares with the G-CSF of 35 Methionins or the single PEGization of 41 Methionins (being met+174 amino acid G-CSF variant) and confirms the former advantage.Standard reductive alkylation reaction in the terminal PEGization G-CSF of N-process has been described among the embodiment 2.Present method can prepare the goods of the basic homogeneous of the terminal PEGization G-CSF of N-. the standard reductive alkylation reaction in the terminal PEGization of total Interferon, rabbit N-has been discussed among the embodiment 3.

Embodiment 1

A. recombinant human met-G-CSF preparation

Recombinant human met-G-CSF (this paper is sometimes referred to as " rh-G-CSF " or " r-met-hu-G-CSF ") is according to the Souza patent, U.S. Patent No., 4,810,643 (this paper quotes for your guidance). the rhG-CSF that method is share be from E.coliIts aminoacid sequence of recombination expression product (by dna sequence encoding) shown in following (SEQ.ID.NO.1 and 2):

ATG?ACT?CCA?TTA?GGT?CCT?GCT?TCT?TCT?CTG?CCG?CAA?AGC?TTT?CTG

M T P L G P A S S L P Q S F L

CTG?AAA?TGT?CTG?GAA?CAG?GTT?CGT?AAA?ATC?CAG?GGT?GAC?GGT?GCT

L K C L E Q V R K I Q G D G A

GCA?CTG?CAA?GAA?AAA?CTG?TGC?GCT?ACT?TAC?AAA?CTG?TGC?CAT?CCG

A L Q E K L C A T Y K L C H P

GAA?GAG?CTG?GTA?CTG?CTG?GGT?CAT?TCT?CTT?GGG?ATC?CCG?TGG?GCT

E E L V L L G H S L G I P W A

CCG?CTG?TCT?TCT?TGT?CCA?TCT?CAA?GCT?CTT?CAG?CTG?GCT?GGT?TGT

P L S S C P S Q A L Q L A G C

CTG?TCT?CAA?CTG?CAT?TCT?GGT?CTG?TTC?CTG?TAT?CAG?GGT?CTT?CTG

L S Q L H S G L F L Y Q G L L

CAA?GCT?CTG?GAA?GGT?ATC?TCT?CCG?GAA?CTG?GGT?CCG?ACT?CTG?GAC

Q A L E G I S P E L G P T L D

ACT?CTG?CAG?CTA?GAT?GTA?GCT?GAC?TTT?GCT?ACT?ACT?ATT?TGG?CAA

T L Q L D V A D F A T T I W Q

CAG?ATG?GAA?GAG?CTC?GGT?ATG?GCA?CCA?GCT?CTG?CAA?CCG?ACT?CAA

Q M E E L G M A P A L Q P T Q

GGT?GCT?ATG?CCG?GCA?TTC?GCT?TCT?GCA?TTC?CAG?CGT?CGT?GCA?GGA

G A M P A F A S A F Q R R A G

GGT?GTA?CTG?GTT?GCT?TCT?CAT?CTG?CAA?TCT?TTC?CTG?GAA?GTATCT

G V L V A S H L Q S F L E V S

TAC?CGT?GTT?CTG?CGT?CAT?CTG?GCT?CAG?CCG?TAA?TAG

Y R V L R H L A Q P * *

(this also is the composition that is used for the non-PEGization of control animal). as selection, also can select for use the Neupogen on the market to carry out following PEGization reaction (this paper quotes its package insert for your guidance).

The preparation of the G-CSF of B.PEGization

The N-bicine N-of 100mM pH8.0, include the solution of the above-mentioned rh-G-CSF of 10mg/ml, joining molecular-weight average is (carboxylic methoxy polyoxyethylene glycol N-hydroxyl succinyl-ester) (Union Carbide) among the 6000 daltonian solid-state SCM-MPEG. the SCM-MPEG is here Duoed 1.5 moles than rh-G-CSF. after stirring 1 hour gently, mixture is diluted to 2mg/ml with sterilized water, and pH transfers to 4.0 with dilute hydrochloric acid.Reaction is at room temperature carried out. and reaction mixture mainly by the single PEGization rh-G-CSF of three kinds of forms, form by the rh-G-CSF of some two PEGization and the rh-G-CSG of unmodified and reaction by-product (N-hydroxyl succinimide) at this moment.

The preparation of the terminal PEGization rh-G-CSF of C.N-

The rh-G-CSF of the single PEGization of three kinds of forms can be separated with ion-exchange chromatography. and reaction mixture is splined on (every milliliter of resin of 1mg protein) Pharmacza SSepharose FF post (Pharmacia XK50/30 storehouse, column volume 440ml), with the buffer A (sodium-acetate of 20mM, pH4.0) balance in addition. clean with 3 times of buffer A then to column volume. protein example is with 15 times of buffer B to column volume (20mM sodium-acetate, pH4.0,1M NaCl), its concentration gradient is carried out the linear gradient wash-out from 0-23%. and wash-out rear pillar bed is cleaned with the buffer A of 3 volumes balance again with the buffer B of equal-volume 100% again. and the flow velocity of whole process should maintain the 8ml/min. elutriant and be monitored at 280nm, with the every pipe fraction collection of 5ml. merge the cut that contains each single PEGization rh-G-CSF kind according to Figure 1A. it is concentrated with band YM1076mm film 350mL Amicon teeter column (stirred cell) that these are merged things.

To carry out sieve chromatography from the amalgamation liquid of ion-exchange chromatography, so that the protein of two PEGization is separated from the protein of single PEGization. in most cases, the mixture upper prop that contains the 5-10mg sample in the 2-5ml solution, post is 120ml Pharmacia Superdex75HR 16/60, with the sodium-acetate of the pH4.0 of 20mM with column equilibration. with 1,5ml/min flow velocity wash-out 100 minutes. with the every pipe fraction collection of 2ml. protein content is added in monitoring at the 280nm place in the elutriant.Merge the cut at different peaks and carry out next step analysis. compare the relative proportion of each place, peak output in the following table.

Table 1

Fractional yield and decorating site

Decorating site is with reference to Figure 1A fractional yield

The terminal peak 1A 3 of N-

Lys-35 peak 2A 2

Lys-41 peak 3A 1

Under this reaction conditions, tangible PEGization reaction may not can take place in 17 and 24 Methionin.

D. feature description

In order to identify the feature of each sample, analyzed from five aspects: (1) SDS-Page (Figure 1B), (2) sieve chromatography HPLC (" SECHPLC ") (Fig. 2), (3) peptide (enzyme is cut) separating spectrum analysis (Fig. 3 A, 3B and 3C), (4) external to G-CSF bioactive test (Fig. 4) and (5) in vivo (in the hamster body) to its bioactive test (Fig. 5 A and 5B).

For each sample composition, the result shows, for the terminal single PEGization G-CSF of N-, surpassed 95% by the ratio of PEGization in the sample, perhaps, remaining is the material (although limit of detection is tested in remaining being lower than in the sample) of not PEGization. as for the percentage composition separately of three kinds of single PEGization products of form (N-end, Methionin 41 and Methionin 35), the result shows, N-end and Methionin 41PEGization product have surpassed 97% of single PEGization gross product, Methionin 35 productive rates of PEGization are lower, may be because its unstable causing in tested condition.

Table 2

The per-cent of the terminal PEGization G-CSF of N-composition

Non-also property SDS PAGE SEC HPLD N-end sequencing *

The G-CSF 97.44 99.43 96.6 of single PEGization

The G-CSF 2.56 0.57 3.4 of unmodified

* the N-end sequencing of discussing below can not be thought quantitative test at this, because when carrying out the N-end sequencing, may cause peg molecule to split away off from protein molecule N-end.

Table 3

The single PEGization product per-cent of three kinds of forms

The terminal PEG-Lys35PEG-of N-Lys41PEG-

GSCF GSCF** GSCF

(RI/UV＝.96)* (RI/UV＝.72) (RI/UV＝1.12)

Irreducibility 97.44 77.4 1100.00

SDS-PAGE

SEC?HPLC 99.43 93.38 99.96

* RI/UV is the index of refracted light and ultraviolet absorption ratio, and it is mainly used to estimate to contain in each protein molecule the molecule number of polyoxyethylene glycol.The index of the ultraviolet absorption of the refraction of application peg molecule and reflection protein molecule is from SEC HPLC data computation.

This form G-CSF of * is unstable under test condition.

Method

1. SDS-PAGEThat .SDS-PAGE use is ISS Daiichi PureChemicals, Co., Tokyo, little glue of the irreducibility 4～20% that Japan produces, and with coomassie brilliant blue staining. gel is scanned with molecule dynamic densitometer (molecularDynamics Densitometer with Image Quant). and the result: .1 swimming lane shown in Figure 1B is (on one side left hand is that a standard molecular weight albumen (Novex Mork 12 molecular weight standard product) .2 road is 3 μ g rh-G-CSF standards; 3 roads are last sample 10 μ gSCM-PEG-GCSF reaction mixture electrophoresis result; That 4 Dao Neijia is the G-CSF of the terminal single PEGization of 10 μ gN-; Be that 10 μ g are from the terminal met number of N-, the G-CSF of the 35th the single PEGization of lysine residue in 5 roads; 6 roads are 10 μ g from the G-CSF. of the 41st the single PEGization of Methionin of N end methionine(Met) from figure as can be seen, contain the single PEGization product of N-end in 3 roads, and demonstrate the wall scroll band.

2. Sieve chromatography-high pressure liquid chromatography

What SEC-HPLC used is Biosep SEC 3000 posts, carries out in water HPLC system, and uses the 100mM sodium phosphate, under the pH6.9 condition, with 1ml/min flow velocity wash-out 20min.The signal at monitoring 280nm place. result: as shown in Figure 2, line " C ", because only contain the rh-G-CSF of the terminal single PEGization of N-, so have only a peak, line " D " (Methionin 35 single PEGization products) is identical with " E " (Methionin 41 single PEGization products) situation. this shows that also single PEGization G-CSF is pure basically in different collection tubes.

3. The peptase cutting is from collection of illustrative plates. use following method, (being called " Mono-PEG-1 ", " Mono-PEG-2 " and Mono-PEG-3 ") is analyzed to three kinds of samples.(a) the single PEGization G-CSF aliquot sample of standard reductive alkylation reaction .500 μ g is carried out rapid vacuum drying, it is mixed with 1mg/950 μ l concentration in the 0.3M Tris-HCl that contains 6M Guanidinium hydrochloride and 1mM EDTA, add iodoacetic acid in the pH of buffer 8.4. sample and carry out the S-carboxymethylation, and 37 ℃ of incubation reaction 20 minutes. sample uses Sephadex G-25 fast rotational albumen sepn post (Quick Spin Protein Columns) to carry out desalting treatment then, back exchange buffering liquid. behind desalination and exchange buffering liquid, sample concentration is diluted to 0.5mg/ml. (b) protein restriction endonuclease SV8 digestion with the damping fluid that increases with it.Sample with SV8 (enzyme-to-substrate than be 1:25) 25 ℃ of digestion process 26 hours. (c) cutting of HPLC peptase is from collection of illustrative plates.The postdigestive protein Vydac C4 post (4.6 * 250mm, particle diameter 5 μ, aperture 300) of packing into carries out wash-out with the linear gradient of acetonitrile among the 0.1%TFA, through the HPLC peptide mapping.The peptide section artificially collects and is dried the usefulness of back in order to order-checking in the rearmounted rapid vacuum drying device (Speed Vac). result: compare with the reference standard sample, (i) (Fig. 3 A) is " Mono-PEG-1 " (be N-terminal single PEGization product), in the time of 57.3 minutes peak value reduce and in the time of 77.5 minutes appearance one new peak; (ii) (Fig. 3 B) is " Mono-PEG-2 ", (Methionin 35PEGization product), and the height at peak descended to some extent and a new elution peak occurred in the time of 66.3 minute in the time of 30.3 minutes; (iii) (Fig. 3 C) is " Mono-PEG-3 " (Methionin 41PEGization product), 30.3 minute the time the peak disappear, and a new elution peak appearred in the time of 66.4 minutes. these peptide sections all demonstrate significant difference in separating spectrum. it can also be seen that because some little incomplete lysates appear in small digestion difference in the both sides of 86.1 minutes peptides from figure. (d) The N-end sequencing is analyzed. each " newly " peptide section all will be identified through the N-terminal sequence analysis in the top collection of illustrative plates. dried peptide section is dissolved in 0.1%TFA, in the ABI protein sequencer, check order. for " Mono-PEG-1 " (N-terminal PEGization product), " newly " peak product of 60% (77.5 minutes time) has carried out 10 round-robin and has checked order.Initial yield 5%, the methionine residues that this means the N-end is by peg molecule. sealing. it should be noted that, resemble this initial peptide and should cause 0 initial yield, here less than 5% value may be since in the sequencing analysis process, cause peg molecule split away off from the terminal methionine(Met) of N-due to .N-terminal peptide fragment sequencing result be: M-T-P-L-G-P-A-S-S. is for " Mono-PEG-2 " (Methionin 35PEGization product), collected and located 80% of peak value cumulative volume in 66.3 minutes, and it to be carried out 9 take turns cycle sequencing. the rate of recovery of Methionin 35 is quite low, mean that PEGization occurs on 35 Methionins. and 41 the Methionin rate of recovery is the same with other amino-acid residue, illustrating that this residue modifies. the ratio of peak respective standard that peptide was located at 30.3 minutes is low with reference to figure, the peak area that peptide was located at 30.3 minutes only is 57.5% of corresponding peptides, to its sequencing result is that K-L-C-A-T-Y-K-L. is for " Mono-PEG-3 " (Methionin 41PEGization product), for the sample of having collected peak cumulative volume 80% at the peptide of 66.4 minutes wash-outs, and it is carried out 9 round-robin check order, the sequence that detects is K-L-C-A-T-Y-K-L, wherein contain 35 consistent with other residue rate of recovery with the rate of recovery of 41 lysine residue .35 position Methionins, the rate of recovery of 41 Methionin is quite low, means that PEGization takes place at this place.The result: " Mono-PEG-1 " is the terminal single PEGization product of N-; " Mono-PEG-2 " is 35 Methionin part PEGization products; " Mono-PEG-3 " is 41 Methionin PEGization products.By compare standard (non-PEGization G-CSF) and the G-CSF1 of single PEGization, 2 and 3 collection of illustrative plates, the two collection of illustrative plates N-terminal peptide peak height of " Mono-PEG-2 " as can be seen (Lys 35) and " Mono-PEG-3 " (Lys 41) all descends slightly to some extent.All mixing in the product of this explanation Methionin 35 and Methionin 41 has minor N-terminal PEG product, and perhaps the methionine(Met) of N-end has small part by PEGization.

4. External activityThis material has activity. and that Fig. 4 shows is the analyzed in vitro result.From scheming as can be seen the rhG-CSF active 68% that the terminal single PEGization product of N-has unmodified.Method: the G-CSF vitro bioassay is the mitogenesis test of share mouse 32D cell G-CSF dependency clone. cell is cultivated with the Iscoves substratum that contains 5% FBS and 20ng/ml rhG-CSF.Before adding sample, cell is with the growth medium flushing that does not contain rhG-CSF 2 times. with 48 to 0.5ng/ml concentration ranges (be equivalent to 4800～50IU/ml) the rhG-CSF typical curves of making 12 points. every duplicate samples preparation estimates to drop on 4 kinds of diluents of (1000 arrive 3000IU/ml) in the typical curve line style part in typical curve trial scope, and with in triplicate. because these samples are obviously lower at external activity, PEGization rhG-CSF sample generally dilutes less than 4～10 times. and the sample of every part 40 μ l volume or standard diluent are added in the suitable aperture of the 96 hole droplet degree plates that contain 10,000 cells/well.At 5.5% CO ₂, cultivate after 48 hours for 37 ℃, every hole add 0.5 μ m Ci methyl- ³Behind H-thymus pyrimidine .18 hour, gather in the crops dull and stereotyped cell and counting, to make dose response curve (logrhG-CSF concentration is to the CPM-background) and the point that drops in the typical curve line style part scope is carried out linear regression analysis. linear equation and dilution factor with gained are proofreaied and correct the concentration of measuring unknown test sample. result: as shown in Figure 4. as can be seen from Figure 4, in three kinds of single PEGization forms, the terminal single PEGization G-CSF of N-is at the high biological activity of external demonstration.

5. Activity in vivoMensuration has also confirmed the activity of the G-CSF of the terminal PEGization of N-in the body. and mensuration is carried out to the sample of the subcutaneous shot 0.1mg/kg of Golden Hamster body weight dosage in the body. and every group of 4 animals are drawn blood through afterbody at each time point. and the blood sample of collection carried out complete blood count the same day, and calculate the white corpuscle average number. from Fig. 5 A and 5B as can be seen, after 1 day appearance effect peak at subcutaneous single injection 0.1mg/kg body weight dosage sample. and two kinds of single PEGization products (N-end and Methionin 35) demonstrate the reaction of prolongation, and (can find out from Fig. 5 B to the rhG-CSF increased activity that the proteinic reaction of Methionin 41PEGization does not demonstrate in vivo than unmodified, activity is also more weaker in fact). these results show that peg molecule can significantly change proteinic result of treatment and the proteinic benefit of PEGization also depends on decorating site adhering on the protein molecule. (the clean average area of the WBC after the single subcutaneous injection under the curve is (according to CRC standard mathematical table, 26th Ed. (Beyer, W.H., Ed.) CRC PressInc., Boca Raton, FL 1981.p.125 calculates) consistent with Lys-35 and the terminal single PEGization product of N-).

E. stability study

In addition, the N-end and the single PEGization G-CSF of the 35 Methionins stability that prepare are above studied. (it is because confirmed the extra activity of the G-CSF that it does not exceed unmodified that 41 Methionin PEGization products are not studied).Unexpectedly other form Methionin 35 single PEGization products than the G-CSF of single PEGization are more stable when storage for the G-CSF that studies show that the terminal PEGization of N-.Stability monitors the product cracking with SEC-HPLC and measures. and terminal PEGization G-CSF of method: N-and Methionin 35 single PEGization G-CSF researchs are finished under 4 ℃ of 2 pH conditions, be pH4.0 and pH6.0,16 days each cycles. it is when making stability study be carried out .pH6.0 quickly that pH is risen to 6.0, terminal single PEGization G-CSF of the N-of above-mentioned gained and Methionin 35 single PEGization G-CSF place and contain the 20mM sodium phosphate, the 5mM sodium-acetate, 2.5% N.F,USP MANNITOL, 0.005%Tween-80, pH are to be that every milliliter of equal portions liquid of 0.25mg/ml. is stored in the 3ml aseptic injection bottle to the protein final concentration in 6.0 the damping fluid. each injection bottle was preserved 16 days in 4 ℃ and 29 ℃.Analyze its stability if draw by SEC-HPLC. measuring result afterwards is consistent (being determined by visual inspection) with initial measuring result (time 0), and then sample is considered to stable in that time.

Result: shown in Fig. 6 A-6C.

(a) compare under 4 ℃ of pH6.0 conditions.Fig. 6 A shows pH6.0, and the SEC-HPLC of the terminal single PEGization G-CSF of N-after through the certain hour section schemes under 4 ℃ of conditions; Fig. 6 B is depicted as Methionin 35 single PEGization G-CSF at pH6.0, under 4 ℃ of conditions through the figure of the SEC-HPLC after the certain hour section. have a kind of explanation to think that in the lysate of Methionin 35PEGization product, the molecular weight that has the just in time G-CSF with unmodified is close.

(b) pH4.0, renewal .pH4.0 under 4 ℃ of conditions, the control condition that provides under 4 ℃ of conditions is the condition of a quite stable, the terminal PEGization G-CSF of N-does not degrade under this condition.For Methionin 35PEGization product, the degraded of product still takes place, but its speed is much slow.

(c) pH4.0, stability under 4 ℃ of conditions is relatively. and Fig. 6 C shows under these conditions, during time limit time expand, the SEC-HPLC figure of the single PEGization product of G-CSSF. from figure as can be seen, at pH6.0, under 4 ℃ of conditions, the Methionin 35 of PEGization when 16 days or 35 days go the increase of PEG level not to be higher than 6 days the time finding level (seeing Fig. 6 B). after this means that PEGization level (unstable) has 6 days, keep under this condition becoming always.

Embodiment 2

Present embodiment has been stated a kind of method of colony of the homogeneous basically that utilizes standard reductive alkylation to react to prepare single PEGization G-CSF, characterized with this colony. the described reorganization of embodiment G-CSF. is as can be seen on this has used, this method not only has superiority aspect the protein of the terminal chemically modified of preparation N-, and the amine key in this standard reductive alkylation reaction process produces more stable in fact product, shown than big-difference as aggregation extent when storing.

A. The single methoxy polyoxyethylene glycol-GCSF that adheres on the terminal alpha-amino group residue of N-connects The preparation of thing.

Preparation rhG-CSF reaction soln (1ml, concentration is 5mg/ml, described in top embodiment) includes the 100mM sodium phosphate, and pH5.0 also contains 20mM NaCNBH ₃, it is fully stirred freezing (4 ℃) after, add mole number and exceed 5 times methoxy polyethylene glycol aldehyde (MPEG) (molecular-weight average is 6kDa). reaction mixture continues to stir under uniform temp.

Proteinic degree of modification is come its monitoring with SEC-HPLC in the reaction process, and what SEC-HPLC used is Bio-Sil SEC 250-5 post (BIO-RAD), under the pH6.8 condition, uses 0.05M NaH ₂PO ₄, 0.05M Na ₂HPO ₄, 0.15M NaCI, 0.01MNaN ₃, damping fluid carries out wash-out with the 1ml/min flow velocity.

SEC HPLC analytical results shows after 10 hours, existing 92% protein transduction becomes the mono-MPEG-GCSF derivative. and this is as can be seen from Figure 7, wherein write down protein concn (recording) with light absorption ratio at the A280 place, the elution peak that has shown the single PEGization G-CSF product that in the time of 8.72 minutes, occurs, the less elution peak of the G-CSF that does not react that occurred at 9.78 minutes.

Fig. 8 has shown the peak with the N-hydroxyl succinyl-fat gained of MPEG as a comparison. its molecular weight also is that 6kDa. is from scheming as can be seen, in reaction mixture, have: tri-MPEG-GCSF connector (peak is takeed on all at 7.25 minutes), di-MPEG-GCSF connector (peak value is at 7.62min), mono-MPEG-GCSF connector (peak value is at 9.87min).

At 10 hours time points, 92% protein was converted to single PEGization product, and the pH of its reaction mixture transfers to pH4.0 with 100mM HCl, and with 1mM HCl reaction mixture was diluted 5 times.

The mono-MPEG-GCSF derivative is that 4.0 equilibrated HiLoad, 16/10 S Sepharose HP post (Pharmacia) carries out purifying with the sodium-acetate buffer pH that ion exchange chromatography share with 20mM.Reaction mixture is with 1ml/min flow velocity dress post, unreacted MPEG carries out wash-out with 3 times of same buffer to column volume. and carry out 400 minutes linear gradient elution then, 20mM sodium-acetate concentration is from 0%-45%, pH4.0, include 1M NaCl, at 4 ℃ of elute proteins-polymkeric substance connector.

The collection tube that will contain the mono-MPEG-GCSF derivative merges, and concentrates and filtration sterilization.

Modify rh-G-CSF and the various forms mono-MPEG-GCSF connector that obtains prepares with similarity method with the MPEG aldehyde of different molecular-weight average (12,20 and 25kDa).

The analysis of B. single PEGization G-CSF

1. molecular weight

Utilize SDS-PAGE, gel-filtration, ground substance assistant laser is resolved mass spectroscopy (matrix assisted laser desorption), and the equilibrium centrifugation method is measured the molecular weight of single PEGization connector. and the result is as shown in table 4 below.

Table 4

N-end alkyl Mono-MPEG-GCSF connector molecular weight

The gel-filtration mass spectrum that connector is estimated is surveyed super centrifugal survey

Molecule measuring weight molecule amount molecular weight

MPEG-

(6kDa)- 24800 53024 24737 25548

G-CSF

MPEG-

(12kDa)- 30800 124343 30703 29711

GCSF

MPEG-

(20kDa)- 38800 221876 38577 38196

GCSF

MPEG-

(25kDa)- 43800 333266 N/D N/D

GCSF

The terminal mono-MPEG-GCSF connector of the N-of preparation structure is cut the figure spectrometry with matter sequential analysis of N-terminal protein and peptase and is confirmed that the terminal bromize fluoride crack with sulphur aminoacyl residue of .N-causes removing of peg molecule.

2. Biological activity

The external biological of PEGization MPEG-GCSF connector is active to stimulate the mouse medullary cell right through measuring ³The absorption of H thymus pyrimidine is measured.

Biological activity is through measuring measuring its total leukocyte number behind MPEG-GCSF connector or rhG-CSF (100mg/ gram body weight dosage) the subcutaneous injection hamster in the body.Compare with the G-CSF of unmodified, the bioactive calculating of PEGization G-CSF is to deduct the vehicle Control area under curve and the expression of the relative reactivity of the .MPEG-GCSF derivative that obtains is described with its percentage activity of comparing with the G-CSF of unmodified with area under the WB/ time curve.

As shown in Figure 9, shown the total leukocyte number reaction of the mono-N-terminal M PEG-GCSF connector that the MPEG aldehyde with different molecular weight (6kDa, 12kDa and 20kDa) is made through the reductibility alkylation reaction. from figure as can be seen, the equal induced reaction of all single PEGization products. except the PEG of 12kDa white corpuscle number after second day white corpuscle number is than the effect of 20kDa PEG product is big slightly, used molecular weight polyethylene glycol is big more, and the white corpuscle number of acquisition can be many more.

3. stability study

Compared extent of polymerization with the terminal PEGization G-CSF of N-of two kinds of different chemical processes (referring to the standard reductive alkylation reaction of amine and acid amides here) preparation. unexpectedly, find that the terminal PEGization G-CSF of the N-that forms with the amine key is much more stable than the terminal PEGization G-CSF of the N-that forms with amido linkage (the NHS method described in the embodiment 1).

Method: the terminal PEGization G-CSF of the N-of two kinds of forms product places the 10mMNaO of pH4.0 _MeIn, wherein contain 5% sorbyl alcohol, the protein molecule final concentration is to contain 1mg protein .G-CSF in every ml solution all with PEG6000PEGization. embodiment 1 is pressed in the preparation of amido linkage connector, amine key connector preparation is undertaken by embodiment 2. and duplicate samples such as 6 of every kind of form are preserved 8 all .8 after week at 45 ℃, measure aggregation extent with sieve chromatography and ion-exchange chromatography.

The result: the result shows that standard reductive alkylation reaction product of the present invention is much more superior than acetylate; because; it is shocking; product is after 8 weeks under the temperature condition that raises, and the former aggregation extent is more much smaller than the latter. and following table is that accumulative percentage ratio does not take place after sieve chromatography (SEC) or ion-exchange chromatography (IE) analysis two kinds of form products separately:

Table 5

45 ℃ of following 8 all % main peak SEC/IE of sample

Amine key product 82%/84%

Amido linkage product 37%/65%*

* this value is higher relatively is because ion-exchange chromatography can not carry out the exhaustive analysis of aggregation extent.

Embodiment 3

This embodiment describes the total Interferon, rabbit of chemically modified. and more particularly, this embodiment has described a kind of method of colony of the homogeneous basically for preparing single PEGization IFN-con1, and the characterized of this colony.

Should be noted that; though that use in the present embodiment is IFN-con1; although but above-mentioned arbitrary total Interferon, rabbit all can be by chemically modified. used polyoxyethylene glycol to modify in the present embodiment; but resembling this chemically modified can carry out with above-mentioned arbitrary water-soluble polymers. turn usefulness into for PEG; though that use in the present embodiment is PEG12000; but other any type of water-soluble PEG all can use (use herein PEG12000 be for be easy to handle and convenient). same; a variety of chemical modification methods (such as acetylization reaction) are also arranged here; but for selective N-terminal chemically modified (as the terminal PEGization reaction of N-), had better be with the reductibility reaction described in this embodiment.

A. the preparation of total Interferon, rabbit

Interferon, rabbit IFN-α con1 (refers to that here IFN-con1 (can be referring to U.S. Patent No. 4,695, it is better than description among 623 Fig. 2 (quote its in full for your guidance) the single PEGization of preparation and has Interferon, rabbit .IFN-con1 and express exogenous DNA and produce in bacterium, and its N-end has a methionyl residue.

B. have the PEGization of Interferon, rabbit

It is 100mM that IFN-con1 solution (3.45mg/ml contains 35.25% N-end closure form) is dissolved in sodium phosphate concentration, and pH4.0 includes 20mM NaCNBH ₃Solution in, abundant mixing after 4 ℃ of coolings adds the methoxy polyethylene glycol aldehyde (MPEG) (molecular-weight average is 12kDa) that exceeds 8 times of molar weights.

Proteinic degree of modification is monitored with reversed-phase HPLC in reaction process, and HPLC adorns post with poly (vinylbenzene/Vinylstyrene), as PLRP-S (PL SeperationSciences Polymer Labaratories).

After 10 hours, the reversed-phase HPLC analytical results shows that the protein transduction that 80% N-end has untight α amino becomes the MPEG-IFN-con1 derivative.

At 10 hours time points, 5 times of reaction mixture dilute with waters, the purifying of mono-MPEG-IFN-con1 derivative is finished with ion exchange chromatography, its used post is a Hiload16/10 S Sepharose HP post (Pharmacia), with the sodium-acetate buffer pH4.0 of 20mM balance in addition. reaction mixture is packed into the flow velocity of 1ml/min in the post, unreacted MPEG aldehyde with 3 times to the same buffer of column volume wash-out in addition. under 4 ℃ of conditions, carry out the wash-out of protein-polymer connector molecule then, 20mM sodium-acetate buffer with 0%～75% carried out linear elution 420 minutes. and pH of buffer is 4.0, contains 1M NaCl.

Merge the cut that contains the mono-MPEG-IFN-con1 derivative, after concentrating, refilter sterilization.

The analysis of the total Interferon, rabbit of C. single PEGization

1. homogeneity

The mono-MPEG-IFN-con1 connector homogeneity of purifying is detected by SDS-PAGE, and what SDS-PAGE used is the prefabricated gradient glue (Integrated Separation System) of 10-20% or 4-20%. and gel shows that mainly being with at molecular weight is the 35kDa place.

What the effective radius of various forms mono-MPEG-IFN-con1 (hydrodynamic radius) was measured usefulness is Superose 6HR 10/30 (Pharmacia) gel-filtration column. at the 280nm place through determination of uv absorption protein .BIO-RAD gel filtration standards as the globular proteins molecular weight marker.

The terminal mono-MPEG-I FN-con1 of the N-of purifying connector structure adopts order-checking of N-terminal protein matter and peptase to cut collection of illustrative plates and is confirmed.

Here must be noted that and contain some N-end closure materials in these IFN-con1 goods, yet PEGization does not take place in them., the material of PEGization is even to be the terminal single PEGization of N-. thereby, in this case, can consider can be with other method with end closure and untight product separately, as with ion exchange chromatography or molecular sieve analytical method.

2. Biological activity

Mono～MPEG-IFN con1 connector external biological activity is to determine through measuring its antiviral biological activity in people (Hela) cell through the external biological of measuring the .mono-MPEG-IFN con1 connector that its antiviral activity measures is active.

Find that (U/mg protein is that unmodified IFN-con1 active 20%. is as foregoing PEGization G-CSF to mono-MPEG (12kDa)-IFN-con1 connector external biological activity, though analyzed in vitro has certain effect to understanding biological activity, but because characteristic slowly discharges, the chemically modified protein matter activity that often records is very low. biological activity is than external height in the body.

D.N-end closure molecule is removed the total Interferon, rabbit of back chemically modified.

Foregoing IFN-con1 also carries out standard reductive alkylation reaction of the present invention after removing N-end closure molecular moiety wherein in advance. described in the above standard reductive alkylation reaction.Used two kinds of molecules of PEG12000 and PEG20000 in the above in the described reductive alkylation process on glycopeptides.

The apparent molecular weight of this molecule is as follows

The SDS-PAG that the connector gel-filtration records records

The apparent molecular weight of apparent molecular weight

monoMPEG(12kDa) 104.0kDa 35.6kDa

IFN-con1

monoMPEG(20kDa) 175.1kDa 55.4kDa

IFN-con1

3 peaks appear among the FPLC ion-exchange chromatogram analysis result of IFN-con1 20kDa PEG connector:

MonoMPEG-IFN-con1: 66% (being eluted to 265.93ml) that accounts for the total area.

Protein aggregate (aggregate) and oligomerization MPEG-IFN-con1 connector: account for the total area 24% (being eluted to 238.42ml) and

Unreacted IFN-con1: account for 10% the total area (being eluted to 328.77ml).

This condition is not further optimized as yet, and available chromatogram or other method are further separated the material of single PEGization.

Although described the present invention, should understand for a person skilled in the art and can change and revise with preferred embodiment.Therefore, in appending claims, attempt to cover all and drop on equivalent variations in the desired scope of the invention.

Sequence table

(1) general information:

(i) applicant: AMGEN INC

The (ii) protein composition and the method for the terminal chemically modified of invention exercise question: N-.

(iii) sequence number: 2

(iv) contact address:

(A) address: Amgen Inc.

(B) street: 1840 Dehavilland Drive

(C) city: Thousand Daks

(D) state: California

(E) country: USA

(F) postal code: 91320

(v) computer-reader form:

(A) medium for storing: diskette

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release #1.0, #1.25. version

(vi) current application materials:

(A) application number:

(B) applying date:

(C) classification:

(viii) proxy/proxy information:

(A) name: Pessin, Karol M.

(B) reference/summary number: A-286

(2) SEQ ID NO:1 information:

(i) sequence signature:

(A) length: 531 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology feature: linear

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:1:

(2) SEQ ID NO:2 information:

(i) sequence signature:

(A) length: 175 amino acid

(B) type: amino acid

(C) chain: strand

(D) topology feature: linear

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:2:

Claims (65)

1. the total Interferon, rabbit of chemically modified partly is connected at least one water-soluble polymers by total ifn protein and forms on partly.

2. the total Interferon, rabbit of the chemically modified of claim 1, wherein said total Interferon, rabbit partly is selected from IFN-con1, IFN-con2 and IFN-con3.

3. the total Interferon, rabbit of the chemically modified of claim 2, wherein said water-soluble polymers be medicinal acceptable.

4. the total Interferon, rabbit of the chemically modified of claim 1, wherein said water-soluble polymers is selected from: dextran, poly (n-V-Pyrol RC), polyoxyethylene glycol, propylene glycol homopolymer, polyoxytrimethylene/ethylene oxide interpolymer, polyoxy ethylization polyvalent alcohol and polyvinyl alcohol.

5. according to the total Interferon, rabbit of the chemically modified of claim 4, wherein said water-soluble polymers partly is a polyoxyethylene glycol.

6. according to the total Interferon, rabbit of the chemically modified of claim 1, wherein said water-soluble polymers part need not be directly connected on the said total Interferon, rabbit part by other linking group.

7. the total Interferon, rabbit of a chemically modified is connected at least one polyalkylene glycol moiety by IFN-con1 and forms.

8.PEG the total Interferon, rabbit of changing.

9. the method for an attached water soluble polymer on total Interferon, rabbit, wherein said water-soluble polymers has single reaction aldehyde radical, and said method comprises:

(a) under the standard reductive alkylation condition,, make total Interferon, rabbit partly and the water-soluble polymers partial reaction enough acid with selectively activate under the pH value of said total Interferon, rabbit part N-terminal alpha-amino group group;

(b) obtain reaction product and

(c) reaction product isolated in the part that can randomly never react.

10. the method for claim 9, wherein said polymkeric substance be medicinal acceptable.

11. the method for claim 9, wherein said water-soluble polymers is selected from: dextran, poly (n-V-Pyrol RC), polyoxyethylene glycol, propylene glycol homopolymer, polyoxytrimethylene/ethylene oxide interpolymer, polyoxy ethylization polyvalent alcohol and polyvinyl alcohol.

12. the method for claim 11, wherein said polymkeric substance is a polyoxyethylene glycol.

13. the method for claim 9, wherein said standard reductive alkylation reaction relates to uses the reductive agent that is selected from sodium borohydride, sodium cyanoborohydride, boric acid dimethylamine, boric acid Trimethylamine 99 and boric acid pyridine.

14. a method of adhering to peg molecule on total interferon molecule, wherein said peg molecule has single reaction aldehyde radical, and said method comprises:

(a) under the standard reductive alkylation condition, under the pH value of the terminal alpha-amino group group of said total Interferon, rabbit, said total Interferon, rabbit and said peg molecule are reacted with selectively activate enough acid;

(b) the total Interferon, rabbit that obtains PEGization with

(c) the total Interferon, rabbit of PEGization can randomly never take place to isolate in the total Interferon, rabbit of PEGization.

15. the method for claim 14, wherein said peg molecule have the molecular weight of about 2kDa to about 100kDa.

16. the PEGization of being produced with the method for claim 14 has the Interferon, rabbit product.

17. the goods of the basic homogeneous of the total Interferon, rabbit of single PEGization.

18. the goods of claim 17 comprise the total Interferon, rabbit of about 90% single PEGization and the total Interferon, rabbit of about 10% not PEGization.

19. medicinal compositions, the goods of homogeneous basically that comprise the total Interferon, rabbit of (a) single PEGization, the total Interferon, rabbit of said single PEGization is connected to total Interferon, rabbit by a polyalkylene glycol moiety and partly goes up and forms, and this connects and passes through the amine key also only at its N-terminal; (b) be less than the total interferon molecule of 5% not PEGization; (c) a kind of medicinal acceptable diluent, adjuvant or carrier gone up.

20. a method for preparing the terminal single PEGization polypeptide of N-, it may further comprise the steps:

(a) make polypeptide that its N-end has α amino in aqueous medium, be enough in standard reductive alkylation and pH acidity react under the condition of selectivity activation α amino with water-soluble polyethylene glycol with an aldehyde radical and

(b) polypeptide of the terminal single Pegylation of separation N-from reaction mixture.

21. the method for claim 20, wherein the molecular weight of employed polyoxyethylene glycol is about 2kDa to 100kDa.

22. the method for claim 21, wherein the molecular weight of employed polyoxyethylene glycol is about 6kDa to 25kDa.

23. each method of claim 20-22, wherein employed polyoxyethylene glycol is a methoxy poly (ethylene glycol) aldehyde.

24. each method of claim 20-23, the reductive agent that wherein uses in standard reductive alkylation is sodium borohydride or sodium cyanoborohydride.

25. each method of claim 20-24, wherein employed polypeptide is total interferon-alpha.

26. the method for claim 25, wherein employed total interferon-alpha is the methionine(Met) part-1 of its N-terminal.

27. each method of claim 20-26, wherein said separating step (b) comprises purifying.

28. the method for claim 27, wherein said purifying comprises the ion exchange chromatography of described reaction mixture and sieve chromatography.

29.N-terminal single Pegylation has interferon-alpha, it obtains by each method of claim 20-28.

30. a medicinal compositions, it comprises the total interferon-alpha of the terminal single Pegylation of N-of claim 29.

31. goods, it comprises the single polymkeric substance/proteic conjugate of significant quantity, this conjugate is made by the hematopoietic proteins with the amino reactive group of one or more effective ε, the polyethylene glycol polymer of wherein said single polymkeric substance/proteic conjugate partly is positioned at N-terminal but not on ε amino, and described polyethylene glycol polymer part is puted together by α amino; And wherein said single polymkeric substance/protein conjugate prolongs with respect to biological activity in the non-body of puting together hematopoietic proteins.

32. the goods of claim 31, wherein said goods comprise the single polymkeric substance/proteic conjugate greater than 80%.

33. the goods of claim 31, wherein said goods comprise at least 90% single polymkeric substance/protein conjugate.

34. the goods of claim 31, wherein said goods comprise greater than 95% single polymkeric substance/protein conjugate.

35. the goods of claim 31, wherein said goods comprise at least 99% single polymkeric substance/protein conjugate.

36. the goods of single Pegylation hematopoietic proteins, described single Pegylation hematopoietic proteins is made by the hematopoietic proteins with the amino reactive group of one or more effective ε, and wherein said polyethylene glycol polymer part mainly is conjugated in the N-terminal of described hematopoietic proteins and puts together by α amino; And wherein said single Pegylation hematopoietic proteins prolongs with respect to biological activity in the non-body of puting together hematopoietic proteins.

37. the hematopoietic proteins goods of claim 31 or 36, the about 2kDa of the molecular weight of wherein said polyoxyethylene glycol is to about 100kDa.

38. the hematopoietic proteins goods of claim 37, the about 6kDa of the molecular weight of wherein said polyoxyethylene glycol.

39. the hematopoietic proteins goods of claim 37, the about 12kDa of the molecular weight of wherein said polyoxyethylene glycol.

40. the hematopoietic proteins goods of claim 37, the about 20kDa of the molecular weight of wherein said polyoxyethylene glycol.

41. the hematopoietic proteins goods of claim 37, the about 25kDa of the molecular weight of wherein said polyoxyethylene glycol.

42. a medicinal compositions, it comprises (a) claim 31,32,33,34,35,36,38,39,40 or 41 goods and (b) pharmaceutically acceptable thinner, carrier or auxiliary.

43. a medicinal compositions, it comprises the goods of (a) claim 37 and (b) pharmaceutically acceptable thinner, carrier or auxiliary.

44.N-the goods of homogeneous basically of the G-CSF of terminal chemically modified or its analogue, optional being present in medicinal upward acceptable diluent, carrier or the adjuvant.

45. the goods of claim 44, wherein said G-CSF carries out chemically modified with the chemical substance that is selected from dextran, poly (n-V-Pyrol RC), polyoxyethylene glycol, propylene glycol homopolymer, polyoxytrimethylene/ethylene oxide interpolymer, polyoxy ethylization polyvalent alcohol and polyvinyl alcohol.

46. the goods of claim 45, wherein said G-CSF or its analogue carry out chemically modified with polyoxyethylene glycol.

47. the goods of claim 46, wherein said polyalkylene glycol moiety partly is connected with G-CSF by amido linkage.

48. the goods of claim 47, wherein the peg molecule that connects by amido linkage is a polyethylene glycol aldehyde.

49. each goods of claim 46 to 48, wherein said G-CSF or its analogue are single Pegylation.

50. the goods of claim 46, wherein said polyoxyethylene glycol have at about 2kDa to the molecular weight between the 100kDa.

51. the goods of claim 50, wherein said polyoxyethylene glycol has about 6kDa to the molecular weight between the 25kDa.

10% the G-CSF that PEGization does not take place or its analogue are formed at the most 52. the goods of claim 44, wherein said goods are reached by the G-CSF of the terminal single PEGization of 90% N-or its analogue at least.

5% the G-CSF that PEGization does not take place or its analogue are formed at the most 53. the goods of claim 52, wherein said goods are reached by the G-CSF of the terminal single PEGization of 95% N-or its analogue at least.

54. the goods of claim 44, wherein said G-CSF has the sequence of identifying in SEQ ID NO.1.

55.N-the goods of the homogeneous basically of the G-CSF of terminal single PEGization, optional being present in medicinal upward acceptable diluent, carrier or the adjuvant, wherein: (a) said G-CSF has the aminoacid sequence of identifying in SEQ ID No.1; (b) the said G-CSF molecular weight single PEGization of polyalkylene glycol moiety that is about 12kDa.

56. a medicinal compositions comprises: (a) goods of the homogeneous basically of the G-CSF of single PEGization, the G-CSF of said single PEGization only are connected on the G-CSF part at the N-of G-CSF end by the amine key by the polyalkylene glycol moiety with the about 12kDa of molecular weight to be formed; (b) less than 5% non-PEGization G-CSF molecule and (c) medicinal acceptable diluent, adjuvant or the carrier gone up.

57. the method for treatment hematopoiesis disorders comprises any one goods of the claim 44-56 of significant quantity on the administering therapeutic.

58. one kind with the method for water-soluble polymers molecule attached on protein or its analogue, wherein said water-soluble polymers has single reaction aldehyde radical, and said method comprises:

(a) under the standard reductive alkylation condition, enough acid, so that the pH value that water-soluble polymers optionally is attached on the said alpha-amino group is following to protein portion and water-soluble polymers partial reaction with the said protein portion N-terminal of selectively activate alpha-amino group group; With

(b) obtain reaction product and

(c) reaction product isolated in the optional part that never reacts.

59. the method for claim 58, wherein said polymkeric substance are medicinal acceptable.

60. the method for claim 58, wherein said water-soluble polymers is selected from: dextran, poly (n-V-Pyrol RC), polyoxyethylene glycol, propylene glycol homopolymer, polyoxytrimethylene/ethylene oxide copolymer, polyoxy ethylization polyvalent alcohol and polyvinyl alcohol.

61. the method for claim 60, wherein said polymkeric substance is a polyoxyethylene glycol.

62. relating to using, the method for claim 58, wherein said reductibility alkylation reaction be selected from sodium borohydride, sodium cyanoborohydride, the reductive agent of boric acid dimethylamine, boric acid Trimethylamine 99 and boric acid pyridine.

63. one kind is attached to method on the G-CSF molecule with peg molecule, wherein said peg molecule contains single reaction aldehyde radical, and said method comprises:

(a) under reductibility alkylation reaction condition,, said G-CSF and said peg molecule are reacted enough acid optionally activation under the pH value of said G-CSF N-terminal alpha-amino group group; With

(b) G-CSF that obtains PEGization with

(c) optionally from non-PEGization G-CSF, isolate PEGization G-CSF.

64. the method for claim 63, wherein said molecular weight polyethylene glycol are that about 6kDa is to about 25kDa.

65. the PEGization G-CSF product of producing with the method for claim 63.

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